Bulletin of Insectology 60 (2): 257-258, 2007 ISSN 1721-8861
Developing a method for phytoplasma identification in cactus pear samples from California Assunta BERTACCINI1, Alberto CALARI1, Peter FELKER2 Dipartimento di Scienze e Tecnologie Agroambientali, Patologia vegetale, University of Bologna, Bologna, Italy 2 D’Arrigo Bross, Salinas, CA, USA
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Abstract Cactus pear plants showing proliferation and stunting of cladodes in Californian cultivations were tested in order to define a molecular methodology for reliable phytoplasma detection. After several unsuccessful trials a simple extraction method was developed to reduce the mucilage content in nucleic acid preparations that was seriously affecting pathogen detection. Nested PCR on 16S ribosomal gene and RFLP analyses together with sequencing of obtained amplicons allow to verify the presence in symptomatic plants of 16SrV-A and 16SrI-B phytoplasmas respectively related to ‘Candidatus Phytoplasma ulmi’ and ‘Ca. P. asteris’. Key words: Cactus pear, nucleic acid extraction, phytoplasma identification, PCR/RFLP, sequencing.
Introduction Cactus pear [Opuntia ficus-indica (L.) Mill.] production is of economic importance in many parts of the world, and in these areas the species shows often symptoms such as stunted growth, cladode enlargement and loss of the green colour that were recently associated with 16SrII-C phytoplasma presence; the disease was also proved to be graft transmissible (Granata et al., 2006). Cladode enlargement was first reported in Mexico but it is also present in Chile, Argentina, South Africa and Italy. Flower proliferation on the flat part of cladodes, premature abscission of flowers and deformation and yellow discoloration of young cladodes have been described in Mexico, South Africa and Italy (Pimienta, 1990; Granata, 1995). Similar symptoms affect some cactus pears in Californian cultivations where severe stunting and cladode proliferation are present. However, in some cases, there is a tendency of recovery in some cladodes of symptomatic plants. But none of the recovered (symptomless) plants bear cladodes of normal size. Thus far, diverse protocols employed for phytoplasma detection in samples collected from these plants have failed. (P. Felker, unpublished data). In the present study a number of different nucleic acid techniques were compared in order to verify presence and identity of phytoplasmas in such ‘fastidious’ plant material. Materials and methods Phloem tissues dissected from cactus pear samples showing stunting and cladode proliferation symptoms and from similar tissues of asymptomatic plants were employed for nucleic acid extraction following four protocols: chloroform/phenol, CTAB (Angelini et al., 2001; de la Cruz et al., 1997) and “DNaesy plant Mini kit Qiagen”. Baby cladodes (2-3 days old) were also collected from the same plants after rooting in greenhouse and nucleic acid was extracted with the same 4
methods described above. Nucleic acid was then maintained at 4 °C and employed at the concentration of 20 ng in direct PCR with universal primers P1/P7 (Deng and Hiruki, 1991; Schneider et al., 1995), followed by nested PCR with primers F1/B6 (Davis and Lee, 1993; Padovan et al., 1995), and by a second nested PCR with R16F2/R2 (Lee et al., 1995). RFLP analyses with selected restriction enzymes, and comparison with phytoplasma reference strains (Bertaccini et al., 2000) was then carried out for identification of associated phytoplasmas. Another extraction procedure was developed to reduce the mucilage content in the preparations by using baby cladodes from greenhouse (see above) and on young fruits from naturally infected plants growing under field conditions. For these experiments 1 g of fresh tissue from phloematic areas of cladodes and of small fruits was ground with liquid nitrogen in sterile mortars with a grinding buffer as described by Lee et al., (1991); after centrifugation at 11,400 rpm for 30 minutes in a Beckman “Allegra 45” centrifuge the supernatant was employed in PCR amplification with the protocol described above after 1 : 30 - 1 : 60 dilution in sterile distilled water. Nested PCR yielded two amplicons, one amplified with nested primer pairs F1/B6 and the other with R16F2/R2. RFLP analyses of the two amplicons were performed. Selected amplicons were purified with “QUIAquick” PCR purification kit and sequenced both directions. Sequences were aligned with those of phytoplasmas available in GenBank and percentage of homology was verified. Results All the four protocols for nucleic acid extraction failed to yield nucleic acid preparations from symptomatic or asymptomatic cactus pear materials that were suitable for PCR amplification. Using the diluted supernatants in PCR, amplicons (putative phytoplasmas 16S rDNA fragments) with ex-
References
Figure 1. RFLP digestion of F1/B6 (left) and R16F2/R2 (right) amplicons from Californian cactus pear. pected length bands were obtained in first nested PCR with F1/B6 primers (about 1,700 bp) and in second nested PCR with R16F2/R2 primer pairs (about 1,200 bp). RFLP analyses with TaqI, RsaI, BfaI and TruI restriction enzymes on F1/B6 (figure 1) and on R16F2/R2 amplicons allow to identify phytoplasmas belonging to ribosomal subgroup 16SrV-A in naturally infected baby cladodes growing in greenhouse and in one of the fruits from the same plant collected in the field. The same phytoplasmas were identified in a fruit collected in field from a different plant showing partial stunting symptoms. A phytoplasma belonging to ribosomal subgroup 16SrI-B was also detected in another fruit collected from naturally infected plants. Sequencing of F1/B6 and of R16F2/R2 amplicons from naturally infected plants and from one of its fruits allow to verify that both sequences share 99% homology with phytoplasmas belonging to ribosomal group 16SrV and share 100% homology between them. Virtual RFLP confirmed the presence of 16SrV-A phytoplasmas i.e. phytoplasmas related to ‘Candidatus Phytoplasma ulmi’ (Lee et al., 2004). Discussion Detection of phytoplasmas in symptomatic cactus pear plants from California confirms that proliferation and stunting symptoms are associated with the presence of phytoplasma. The finding of phytoplasmas distinct from 16SrII-C previously reported to be associated with this disease of cactus pears, indicates that diverse phytoplasmas can be associated in cactus pear with similar symptoms. Moreover, this is the first report of identification of an elm yellows phytoplasma in cactus pear, while aster yellows phytoplasmas (16SrI-B) were recently described in one symptomatic plant of Opuntia sp. from a village of Yunnan region in China (Wei et al., 2007). Further improvement of the extraction methodology will allow to extensive survey in Californian fields to reduce the disease impact in cactus pear cultivations.
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ANGELINI E., CLAIR D., BORGO M., BERTACCINI A., BOUDONPADIEU E., 2001.- Flavescence dorée in France and Italy: occurrence of closely related phytoplasma isolates and their near relationships to Palatinate grapevine yellows and an alder yellows phytoplasma.- Vitis, 40: 79-86. BERTACCINI A., CARRARO L., DAVIES D., LAIMER DA CAMARA MACHADO M., MARTINI M., PALTRINIERI S., SEEMÜLLER E., 2000.- Micropropagation of a collection of phytoplasma strains in periwinkle and other host plants, p. 101. In: 13th International Congress of IOM, July 14-19, Fukuoka, Japan. DAVIS R. E., LEE I.-M., 1993.- Cluster-specific polymerase chain reaction amplification of 16S rDNA sequences for detection and identification of mycoplasmalike organisms.Phytopathology, 83: 1008-1001. DE LA CRUZ M., RAMIREZ F., HERNANDEZ H., 1997. -DNA isolation and amplification from cacti.- Plant Molecular Biology Reporter, 15: 319-325. DENG S., HIRUKI C., 1991.- Genetic relatedness between two non-culturable mycoplasmalike organisms revealed by nucleic acid hybridyzation and polymerase chain reaction.Phytopathology, 81: 1475-1479. GRANATA G., 1995.- Biotic and abiotic diseases. Agroecology, cultivation and uses of cactus pear.- FAO Plant Production and Protection, 132: 109-119. GRANATA G., PALTRINIERI S., BOTTI S., BERTACCINI A., 2006.Aetiology of Opuntia ficus-indica malformations and stunting disease.- Annals of Applied Biology, 149: 317-325. LEE I.-M., DAVIS R. E., HIRUKI C., 1991.- Genetic interrelatedness among clover proliferation mycoplasmalike organisms (MLOs) and other MLOs investigated by nucleic acid hybridization and restriction fragment length polymorphism analyses.- Applied Environmental Microbiology, 57: 35653569. LEE I.-M., BERTACCINI A., VIBIO M., GUNDERSEN D. E., 1995.Detection of multiple phytoplasmas in perennial fruit trees with decline symptoms in Italy.- Phytopathology, 85: 728735. LEE I.-M. MARTINI M., MARCONE C., ZHU S. F., 2004.Classification of phytoplasma strains in the Elm yellows group (16S rV) and proposition of ‘Candidatus Phytoplasma ulmi’ for the phytoplasmas associated with elm yellows.- International Journal of Systematic and Evolutionary Microbiology, 54: 337-347. PADOVAN A. C., GIBB K. S., BERTACCINI A., VIBIO M., BONFIGLIOLI R. E., MAGAREY P. A., SEARS B. B., 1995.Molecular detection of the Australian grapevine yellows phytoplasma and comparison with a grapevine yellows phytoplasma from Emilia-Romagna in Italy.- Australian Journal of Grape Wine Research, 1: 25-31. PIMIENTA B. E., 1990.- El Nopal Tunero.- Grafica Nueva, Pipila 638. Guadalajara, Jalisco, Mexico. SCHNEIDER B., SEEMÜLLER E., SMART C. D., KIRKPATRICK B. C., 1995.- Phylogenetic classification of plant-pathogenic mycoplasma-like organisms or phytoplasmas, pp. 369-380. In: Molecular and diagnostic procedures in mycoplasmology, vol. 2 (RAZIN S., TULLY J. G., Eds).- Academic Press, New York, USA. WEI W., CAI H., CHEN H., DAVIS R. E., ZHAO Y., 2007.- First report of natural infection of Opuntia sp. by a ‘Candidatus Phytoplasma asteris’- related phytoplasma in China.- Plant Disease, 91: 461. Corresponding author: Assunta BERTACCINI (e-mail:
[email protected]), DiSTA, Patologia vegatale, viale Fanin 42, 40127 Bologna, Italy.
